[1]晁荣荣,郑柳,范晶,等.胰岛素样生长因子1对人RPE细胞分泌TGF-β2、MMP-2的影响及机制研究[J].眼科新进展,2024,44(7):512-517.[doi:10.13389/j.cnki.rao.2024.0098]
 CHAO Rongrong,ZHENG Liu,FAN Jing,et al.Effects and mechanism of insulin-like growth factor 1 on the secretion of transforming growth factor β2 and matrix metalloproteinase 2 in human retinal pigment epithelial cells[J].Recent Advances in Ophthalmology,2024,44(7):512-517.[doi:10.13389/j.cnki.rao.2024.0098]
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胰岛素样生长因子1对人RPE细胞分泌TGF-β2、MMP-2的影响及机制研究/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
44卷
期数:
2024年7期
页码:
512-517
栏目:
实验研究
出版日期:
2024-07-01

文章信息/Info

Title:
Effects and mechanism of insulin-like growth factor 1 on the secretion of transforming growth factor β2 and matrix metalloproteinase 2 in human retinal pigment epithelial cells
作者:
晁荣荣郑柳范晶丁芝祥
541100 广西壮族自治区桂林市,桂林医学院(晁荣荣,范晶);541001 广西壮族自治区桂林市,桂林医学院附属医院(郑柳,丁芝祥)
Author(s):
CHAO Rongrong1ZHENG Liu2FAN Jing1DING Zhixiang2
1.Guilin Medical University,Guilin 541100,Guangxi Zhuang Autonomous Region,China
2.Affiliated Hospital of Guilin Medical University,Guilin 541001,Guangxi Zhuang Autonomous Region,China
关键词:
近视视网膜色素上皮细胞胰岛素样生长因子1磷脂酰肌醇-3-激酶/蛋白激酶B通路转化生长因子β2基质金属蛋白酶2
Keywords:
myopia retinal pigment epithelial cells insulin-like growth factor 1 phosphoinositide 3-kinase/protein kinase B signaling pathway transforming growth factor β2 matrix metalloproteinase 2
分类号:
R778.1
DOI:
10.13389/j.cnki.rao.2024.0098
文献标志码:
A
摘要:
目的 研究胰岛素样生长因子1(IGF-1)对人视网膜色素上皮细胞(ARPE-19)表达转化生长因子β2(TGF-β2)、基质金属蛋白酶2(MMP-2)的影响,并探索其作用机制。
方法 ARPE-19细胞分别按不同浓度IGF-1和不同浓度LY294002培养6 h、12 h、24 h、48 h,采用CCK-8法检测细胞活力,确定IGF-1、LY294002的最佳作用浓度与时间。细胞划痕法检测细胞迁移活性。ELISA法检测细胞培养上清液中TGF-β2浓度。将ARPE-19细胞分为对照组、IGF-1组(80 μg·L-1 IGF-1)、IGF-1+LY294002组(80 μg·L-1 IGF-1+30 mmol·L-1 LY294002)、LY294002组(30 mmol·L-1 LY294002),使用无血清DMEM/F12培养基培养,对照组不做任何处理,分别采用RT-PCR、Western blot检测细胞中TGF-β2、MMP-2、磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(AKT)的mRNA和蛋白表达量。
结果 与0 μg·L-1 IGF-1比较,80 μg·L-1 IGF-1的细胞活力24 h变化显著(P<0.05),故确定其为 IGF-1最佳作用浓度和时间。与0 mmol·L-1 LY294002比较,24 h的30 mmol·L-1 LY294002接近半数抑制浓度,故确定其为LY294002最佳作用时间和浓度。细胞划痕法检测结果显示,0 μg·L-1 IGF-1组、40 μg·L-1IGF-1组、80 μg·L-1 IGF-1组细胞迁移率整体比较及两两比较差异均有统计学意义(均为P<0.05)。ELISA检测结果显示,0 μg·L-1 IGF-1组、40 μg·L-1 IGF-1组、80 μg·L-1 IGF-1组细胞上清液中TGF-β2浓度整体比较及两两比较差异均有统计学意义(均为P<0.05)。RT-PCR、Western blot检测结果显示,IGF-1、LY294002培养24 h,与对照组比较,IGF-1组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均升高,而LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05);与IGF-1组比较,IGF-1+LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05)。
结论 IGF-1能促进ARPE-19细胞增殖、迁移;IGF-1可能通过PI3K/AKT信号通路上调ARPE-19细胞中TGF-β2、MMP-2的表达,参与近视的发生与发展。
Abstract:
Objective To investigate the effects of insulin-like growth factor 1 (IGF-1) on the expression of transforming growth factor β2 (TGF-β2) and matrix metalloproteinase 2 (MMP-2) in human retinal pigment epithelial cells (ARPE-19) and related mechanisms.
Methods ARPE-19 cells were cultured for 6 h, 12 h, 24 h and 48 h, respectively, with different concentrations of IGF-1 and LY294002. The cell viability was detected using the cell counting kit-8 to determine the optimal action concentration and time of IGF-1 and LY294002. The cell migration activity was detected using the cell scratch assay. The concentration of TGF-β2 in cell culture supernatant was detected using the enzyme-linked immunosorbent assay (ELISA). ARPE-19 cells were divided into the control group, IGF-1 group (80 μg·L-1 IGF-1), IGF-1+LY294002 group (80 μg·L-1 IGF-1+30 mmol·L-1 LY294002), and LY294002 group (30 mmol·L-1 LY294002) and cultured with serum-free DMEM/F12 medium, while cells in the control group received no treatment. The mRNA and protein expression levels of TGF-β2, MMP-2, phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) in the cells were measured using the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively.
Results Compared with the 0 μg·L-1 IGF-1 group, the cell viability in the 80 μg·L-1 IGF-1 group changed the most significantly at 24 h (P<0.05); thus, the optimal concentration of IGF-1 was 80 μg·L-1 and the optimal culture time was 24 h. Compared with the 0 mmol·L-1 LY294002 group, the inhibition concentration in the 30 mmol·L-1 LY294002 group at 24 h was close to half; thus, the optimal concentration of LY294002 was 30 mmol·L-1 and the optimal culture time was 24 h. The cell scratch assay results showed that the cell migration rate was significantly different among the 0 μg·L-1 IGF-1 group, 40 μg·L-1 IGF-1 group, and 80 μg·L-1 IGF-1 group (all P<0.05). ELISA results showed that there was a statistically significant difference in the concentration of TGF-β2 in cell supernatant among the 0 μg·L-1 IGF-1 group, 40 μg·L-1 IGF-1 group, and 80 μg·L-1 IGF-1 group (all P<0.05). RT-PCR and Western blot results showed that after 24 h culture with IGF-1 and LY294002, compared with the control group, the mRNA and protein expression levels of TGF-β2, MMP-2, PI3K and AKT in the IGF-1 group increased, while the mRNA and protein expression levels of TGF-β2, MMP-2, PI3K and AKT in the LY294002 group decreased (all P<0.05). Compared with the IGF-1 group, the mRNA and protein expression levels of TGF-β2, MMP-2, PI3K and AKT in the IGF-1+LY294002 group decreased (all P<0.05).
Conclusion IGF-1 can promote the proliferation and migration of ARPE-19 cells. IGF-1 may up-regulate the expression of TGF-β2 and MMP-2 in ARPE-19 cells through the PI3K/AKT signaling pathway and participate in the occurrence and development of myopia.

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备注/Memo

备注/Memo:
国家自然科学基金项目(编号:82160197)
更新日期/Last Update: 2024-07-05